Lateral transfer of a lectin-like antifreeze protein gene in fishes
Introduction Fishes that live in the icy seawater of arctic environments are most commonly protected from freezing by endogenous antifreeze proteins (AFPs) that bind ice crystals and prevent their growth. A number of AFPs have evolved over the last 20 million years of evolution evolution. The distribution of different types of AFPs is puzzling--three very similar type II AFPs are observed in three distantly related species (herring, smelt, and sea raven) that cannot be explained by convergent evolution. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences and high identity regions in their genes. The next closes homolog showed only 40% amino acid sequence identity. Further, genomic Southern blots demonstrated that the lectin-like AFP gene of interest was absent from all other fish species tested. The conservation of the genetic sequences and lack of correlation between evolutionary distance and mutation rate make it unlikely that the gene for this AFP was passed down vertically via evolution (1). The common structural theme between the three type II AFPs and their distinguishing feature among C-type lectin domains is their ten cysteines forming five disulfide bridges in identical positions. The majority of C-type lectins have either two or three disulfide bridges. The independent gain of three disulfide bridges in precisely the same location at three different points in evolution seems unlikely, indicating horizontal gene transfer as the means by which these very different species acquired these very similar AFPs. Methods Isolation of genomic and gene-specific DNAs Genomic DNAs were isolated from the testes, liver, muscle, or whole fish of a number of different fish species from all over the world. Rainbow trout from Denmark, zebrafish from the local pet store, bowfin, yellow perch and cisco from Lake Ontario, Pacific herring from the Pacific coast of Canada, and herring from the north Atlantic. Primers were used to amplify the AFP gene sequences and 16s RNA sequences characteristic of evolution. Phylogenetic analyses The evolutionary affinities of the AFPs were inferred using the Bayesian and parsimony approaches on the amino acid alignment. Human pancreatic stone protein (PSP) was used as an out-group for both. Bioinformatic analyses Databases searches were conducted with the complete sequences of rainbow smelt, herring, and sea raven AFPs. Additionally, protein sequences were used in both protein-protein, position-specific iterated and translated BLAST searches depending on the database searched. cDNA and genomic sequences were used in BLAST searches. Discussion The isolated occurrence of type II AFP in three distant branches of the teleost radiation is virtually unprecedented. The type II AFPs of interest exhibit unique disulfide bridge characteristics and are far more similar to each other than to any other lectin homolog. Their resemblance extends to the DNA sequence level. Even the introns segments of the DNA are up to 97% identical. The best explanation for the up to 85% amino acid sequence similarity, low mutation rate, and extreme conservation intron segments is that type II AFP gene has been laterally transferred. There are a number of possible mechanisms that could explain LGT between species of fish. Among these are genetic transfer by shared parasites, viruses or transposable elements. Another, simpler explanation is sperm-mediated LGT. Sperm-mediated LGT is based on the ability of sperm to absorb foreign DNA from solution. Partial uptake of DNA by the sperm nucleus has been observed for many species, including zebrafish. Transgenic offspring has been generated via sperm-mediated LGT for a variety of species ranging from bees and sea urchins to fish, birds, mammals and other vertebrates. The exogenous DNA usually persists extrachromosomally for some time, but chromosomal integration has been observed in certain cases, such as with the fish, Labeo rohita. References 1. Graham, L. A., Lougheed, S. C., Ewart, K. V., & Davies, P. L. (2008). Lateral transfer of a lectin-like antifreeze protein gene in fishes. PLoS One, 3(7), e2616. 2. Ohno S (1970) Evolution by gene duplication. Heidelberg: Springer-Verlag.